Ca2+ dependent arrhythmias underlie or contribute to sudden cardiac death (SCO). Project 3[unreadable] of the Marks Program Project Grant application seeks to investigate the subcellular basis of such[unreadable] arrhythmogenesis. Experiments are proposed to characterize quantitatively the cellular, subcellular, and[unreadable] molecular triggers that initiate fatal cardiac arrhythmias. Specifically Ca2* sparks and other cellular and[unreadable] subcellular Ca2+ signals will be measured in mouse models of Ca2+ dependent arrhythmogenesis.[unreadable] Preliminary data from the PI and other members of the PPG team suggests that increased sensitivity of the[unreadable] cardiac ryanodine receptor (RyR2), alterations in SR Ca2+ content, and changes in RyR2 macromolecular[unreadable] complex can each contribute to this process. Our approach enables us to investigate the central hypothesis[unreadable] of the PPG: SCO can be caused by disruption of molecular complexes and processes that, in normal hearts,[unreadable] underlie balanced regulation of cellular activity.[unreadable] Using patch clamp methods with confocal Ca2+ imaging at high temporal and spatial resolution, Ca2+[unreadable] sparks and Ca2+ signals will be measured in single mouse ventricular myocytes. In some experiments[unreadable] intracellular sodium concentration, [Na+]i, will be measured. Additionally, step changes in intracellular Ca2+[unreadable] will be imposed using a new flash photolysis method with improved resolution, sensitivity, and control.[unreadable] Mouse models will be used to investigate two important interrelated questions. (1) How does modulation of[unreadable] the cardiac ryanodine receptor (RyR2) account for significant changes in cardiac Ca2+ signaling? (2) Can the[unreadable] properties of sarcolemmal ion channels contribute to pathological remodeling of cardiac Ca2+ signaling to[unreadable] produce a significant proarrhythmic substrate? Preliminary experiments from each of the three tightly linked[unreadable] projects of this PPG suggest that the planned experiments are likely to reveal novel regulatory elements in[unreadable] the control of subcellular Ca2+ signaling. The planned work should lay the foundation for improved[unreadable] understanding of Ca2+ signaling in SCO and provide the insight needed to devise novel molecular-targeted[unreadable] therapeutic strategies for treatment.